Here we plot the results of the metabolisation assay in liquid cultures of Microbacteria from the maize root bacteria (Thoenen et al. 2023) and the AtSphere collection (Bai et al. 2015). We tested MBOA and DIMBOA-Glc. Additionally we also report growth data of all Microbacteria in MBOA-containing half-strength TSB medium to test them for MBOA tolerance and in MBOA-containing minimal medium to test them for using MBOA as sole carbon source for growth.

MBOA metabolisation

Quantification & calculations: using a standard curve the samples are quantified

Prepare standard curves

Calculate the concentrations

Quick overview of measured/detected BXs

Phylogenetic tree

Prepare tree with information on host origin

Add AMPO phenotype on plates

Add quantitative MBOA & AMPO phenotypes

Prepare orthogroup data for the 5 OGs that show 100% sensitivity and specificity for AMPO phenotype on plate

Plot AMPO phenotype in plate assay along with quantitative MBOA and AMPO concentrations measured for liquid cultures grown in half-strength TSB supplemented with 500 uM MBOA. Orthogroups showing 100% sensitivity and specificity are shown on the side. Note that KHB019 was not assessed in liquid culture and therefore is shaded in grey for MBOA and AMPO.

Figure S8a: BX compounds measured after incubation in half-strength TSB supplemented with 500 uM MBOA.

DIMBOA-Glc metabolisation

Quantification & calculations: using a standard curve the samples are quantified

Prepare standard curves

join 11 stds and HMPAA

remove the compounds which are not reliable quantified

color code Compounds

Dilutions: 150 + 350 = 500 50 + 700 = 750 500 / 150 = 3.33333 750 / 50 = 15

1/((500/150)*15) = 0.02

for some reason the MBOA concentrations are a bit too high (factor 1.273141 controlled with t0 MBOA sample) –> ev because of precipitaion issue?

Explore raw data

Phylogenetic tree

Figure S8b: BX compounds measured after incubation in half-strength TSB supplemented with DIMBOA-Glc

Metabolite categories for comparative genomics

MBOA

MBOA metabolisation
max_MBOA max_AMPO max_HMPAA
516.2433 302.6399 44.06674

DIMBOA-Glc metabolization

DIMBOA-Glc metabolisation
max_DIMBOA_Glc max_MBOA max_AMPO
575.2392 120.9501 7.355668

Groups

Growth Microbacteria TSB (Metabolite samples) & MM (Carbon source)

Growth curves of all plates single treatments

AUC

To quantify the total bacterial growth over time, we calculate the total area under the curve. This is done with the function “auc()”. Before that, negative values for density increase were filtered out (i.e., strains or replicates that didn’t grow). For comparison between treatments, the total AUC (calculated using density increase) and AUC_raw (calculated using raw density) is normalized with the AUC of the strain grown in the control treatment (no chemicals added, just normal growth media with DMSO).

TSB

Minimal media

Plotting growth curves of minimal media

3 replicates (Rep) of each strain per plate 2 replicates (Replicate) per plate (twice the identical plate per run) 2 identical runs

Phylogenetic tree

Fig 8c: Growth of strains in minimal medium with 500 uM MBOA.

Binary interpretation of bacterial growth:
For binary interpretation (Bacterium X uses MBOA as C-source: YES or NO) of bacterial growth (quantitatively shown in Fig. S8c), we compared the AUC[MBOA] vs. AUC[DMSO]. The binary data is then reported in Fig. 3 (“C-Source”). We defined a cutoff of >1.5x for the AUC[MBOA]-AUC[DMSO] for bacterial growth on MBOA as sole carbon source.

Figure S8c

Combined tree

Thresholds for qualitative analysis - weak MBOA-degraders: >30% of MBOA degraded compared to the control) - strong MBOA-degraders: (>90% of MBOA degraded, Fig. 3a). strong AMPO-formers (100 - 10 %) weak AMPO-formers’, <10% of max. AMPO forming strain) (0.01 µM, limit of detection).

  1. Tree with AMPO Phenotype on plates

  2. Tree as above with MBOA metabolites

  3. Tree as above with DIMBOA-Glc metabolites

  4. Tree as above with carbon source data

  5. Tree as above with orthogroups

Figure 3 note: phenotype of KHB019 on plate was adjusted in Illustrator to no AMPO.

## R version 4.3.3 (2024-02-29)
## Platform: aarch64-apple-darwin20 (64-bit)
## Running under: macOS Sonoma 14.5
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## Matrix products: default
## BLAS:   /Library/Frameworks/R.framework/Versions/4.3-arm64/Resources/lib/libRblas.0.dylib 
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## attached base packages:
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## other attached packages:
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